Floating aggregates of FLC cells, referred to as FLC organoids, were prepared and cultured as described previously[20 ] from the xenografts established using the patient‐derived FLC tumor line transplanted in 3‐month‐old female NOD‐scid IL2Rgammanull mice (Jackson Laboratory) and were incubated in Kubota's Media (PhoenixSongs Biologicals) supplemented with 5 μm PRI‐724 (β‐catenin inhibitor; Selleck Chemicals) or DMSO for 24, 48, or 96 h. The protocol was approved by the Institutional Animal Care and Use Committee at CCHMC. Total RNA was extracted from FLC organoids using the ReliaPrep RNA Tissue Miniprep System (Promega). Poly‐A selected RNA was reverse‐transcribed using the Illumina TruSeq stranded mRNA library preparation kit and sequenced using Illumina NovaSeq 6000 (paired‐end 100 bp). Raw reads were aligned with the reference genome (GRCh38) using HISAT2,[21 ] and transcript/gene abundance was determined using Kallisto with annotations provided by University of California Santa Cruz using default parameters settings.[22 ] All detected transcripts were tested for differential expression using a moderated t test with a cutoff false discovery rate < 0.05. Fusion transcripts identified in each sample using FusionCatcher[23 ] along with regular transcripts were used to build an index in Kallisto.[22 ] Differential transcript expressions were assessed with the R package DESeq2.[24 ]
Il2rgammanull mice
Manufactured by Jackson ImmunoResearch
IL2Rgammanull mice are a genetically modified mouse model that lacks the interleukin-2 receptor gamma (IL2Rγ) gene. This gene is essential for the development and function of various immune cells, including T cells, B cells, and natural killer cells. These mice are commonly used in research to study the role of IL2Rγ in immune system development and function.
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4 protocols using il2rgammanull mice
1
Transcriptomic Analysis of FLC Organoids
2
Orthotopic Xenograft Mouse Model for Brain Tumors
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GSC-0131 cells were infected with pGIPZ-shRNA virus and selected for 3 days in puromycin (2μg/mL), such that > 80% of cells were GFP+. Cells were then harvested using Accutase (Sigma), counted, resuspended in an appropriate volume of culture media, and kept on ice prior to immediate transplantation. NOD-scid IL2Rgammanull mice (Jackson Labs #005557) were anesthetized by IP injection of 0.2ml/10 grams 1.25% Avertin Solution and kept at 37°C. A small bore hole was made in the skull using a hand drill with a Meisinger #009 steel burr bit (Hager & Meisinger GmbH). 2×10^5 cells were slowly injected by pipet into the right frontal cortex approximately 2mm rostral to Bregma, 2mm lateral and 3mm deep through a 0.2-10ul disposable sterile aerosol barrier tip (Fisher Scientific #02-707-30). The burr hole was closed using SURGIFOAM (Johnson & Johnson) and the skin rejoined using TISSUMEND II (Veterinary Product Laboratories, Phoenix AZ). Seven weeks after initial transplantation mice were injected intravenously with 50μl of 40μM Chlorotoxin: Cy5.5 conjugate [57 (link)] 2 hours prior to sacrifice by carbon dioxide inhalation. The brain and tumor were removed from the skull and imaged for Cy5.5 and GFP fluorescence using the Xenogen IVIS Spectrum imaging system (Caliper Life Sciences).
3
In vivo Verification of Cisplatin-Resistance
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To verify Cisplatin-resistance in vivo, first, the cultured SCLC tumor cells from 1-year Cisplatin-treated mice were implanted as before into nude mice and subjected to the same Cisplatin treatment protocol. Second, the resistance of the cultured SCLC tumor cells was compared with that of the parental cells H69 in the same mouse; Briefly, Female NOD-scid IL2Rgammanull mice (Stock # 055557, Jackson Lab), 10-to-12 weeks old, were used for the second resistance verification test. An equal number (107 cells/ mouse) of cultured SCLC tumor cells and parental H69 cells were implanted s.c. into SCID mice, separately (n=5). I.v. injection of Cisplatin (10mg/kg/week) started when palpable, significant tumor lumps were observed on both flanks. Tumor volume and body weight were measured twice a week. Tumor volumes were calculated using the greatest longitudinal diameter (length) and the greatest transverse diameter (width), as measured by external caliper. The modified ellipsoidal formula was used to express the tumor volume = 1/2(length × width2) [45 (link)] [46 (link)].
4
Xenograft Tumor Imaging in Mice
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